The goal of this project is to provide a comprehensive description of human subtelomeric DNA structure. A complete analysis of the DNA sequence content and organization of human subtelomeric regions is essential for understanding their structure, evolution, and function. Distal telomere regions have been shown to have direct roles in the control of cellular proliferation. In lower eukaryotes, subtelomeric gene expression can be affected both by general proximity to the molecular telomere and by specific features of the sequence organization of subtelomeric loci. A complete description of human subtelomeric structure is required to test hypotheses invoking telomere variation as a regulatory feature of cellular proliferation, to ascertain which human genes might be affected by telomeric position, and to provide telomeric closure for the complete nucleotide sequence maps being constructed as part of the Human Genome Initiative. The complexity and atypical organization of subtelomeric DNA sequences complicate conventional global strategies for very high-throughput DNA sequencing. A focused effort is therefore required to obtain and verify the nucleotide sequences and long-range sequence organization of these regions. Work carried out in the initial 5 years of this project has resulted in the isolation of probes and cloned fragments representing the distal-most 200 kb to 300 kb segments of most human telomeres. The proposed experiments are designed to complete the high-resolution structural analyses of most of these key chromosome regions, integrating the (collaborative) generation of the bulk of subtelomeric DNA sequence data with gap-filling, closure, and confirmation of final sequence organization using RARE cleavage mapping and long-range PCR methods. Confirmation of genomic sequence organization (and consequence detection of subtelomeric variation, where present) is a critical parameter for judging the quality of subtelomeric sequence data, and is a key feature of the strategy for the detailed structural analysis of these regions.